SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Plasmids
2.2. Syncytia Imaging
2.3. Quantitative Cell-to-Cell Fusion Assay.
2.4. Surface Biotinylation
2.5. Antibodies and Immunoblots.
2.6. Generation of Pseudotyped Particles
2.7. Pseudotyped Entry Assay
2.8. S Incorporation Detection
2.9. Recovery of rVSV-CoV2-S
2.10. Viral Replication Curves
2.11. Live-Cell Luciferase Assay
2.12. Specific Infectivity Determination
2.13. rVSV/SARS-CoV2 Antibody Neutralization Assay
2.14. rVSV/SARS-CoV2-S Entry and Cell Viability Assays with Inhibitors
3. Results
3.1. Trimming of the SARS-CoV-2 S Cytoplasmic Tail Enhances Cell-Cell Fusion.
3.2. Truncations in the Cytoplasmic Tail Do Not Necessarily Increase S Protein Levels on the Cell Surface.
3.3. S Cytoplasmic Tail Deletions Enhance ppVSV Transduction Efficiency
3.4. rVSV/SMet1D614G∆21 Recombinant Virus Produces Relatively Low Titers with Poor Specific Infectivity
3.5. rVSV/SMet1D614G∆21 Can Be Used to Characterize Cellular Requirements for Entry
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Havranek, K.E.; Jimenez, A.R.; Acciani, M.D.; Lay Mendoza, M.F.; Reyes Ballista, J.M.; Diaz, D.A.; Brindley, M.A. SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus. Viruses 2020, 12, 1465. https://doi.org/10.3390/v12121465
Havranek KE, Jimenez AR, Acciani MD, Lay Mendoza MF, Reyes Ballista JM, Diaz DA, Brindley MA. SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus. Viruses. 2020; 12(12):1465. https://doi.org/10.3390/v12121465
Chicago/Turabian StyleHavranek, Katherine Elizabeth, Ariana R. Jimenez, Marissa Danielle Acciani, Maria Fernanda Lay Mendoza, Judith Mary Reyes Ballista, Darren Austin Diaz, and Melinda Ann Brindley. 2020. "SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus" Viruses 12, no. 12: 1465. https://doi.org/10.3390/v12121465